Small interfering RNAs (siRNA) in lipid complexes applied intravaginally induce durable gene silencing[unreadable] lasting for at least 9 days throughout the epithelium, lamina propria and stroma of mouse genital tissue, but[unreadable] not in distal organs. Topical application of siRNAs targeting herpes simplex virus type 2 (HSV-2) protects[unreadable] mice from lethal vaginal viral challenge. The goal of this project is to use mouse models to build on these[unreadable] promising preliminary data to develop an siRNA-based topical microbicide for HIV. This project will take[unreadable] advantage of the low cost and ease of manipulation of mouse models to examine the optimal siRNA[unreadable] formulation suitable for vaginal application and examine toxicity and pharmacokinetics at different stages of[unreadable] the menstrual cycle. Although the effectiveness of an siRNA-based microbicide to block HIV transmission[unreadable] cannot be tested in mice, the effect of varying siRNA formulations on inhibiting HSV-2 transmission will be[unreadable] evaluated. Since HSV-2 infection is an important cofactorfor HIV transmission, information about blocking[unreadable] HSV-2 in mice may be directly relevant to designing an ultimate formulation targeting both viruses. However,[unreadable] HSV-2 infects epithelial and neuronal cells, while HIV transmission is likely via Langerhans cells (LC) and[unreadable] lamina propria T cells, dendritic cells (DC) and macrophages. Therefore, HSV-2 protection does not[unreadable] necessarily translate into protection against HIV transmission. To achieve this goal, silencing must be[unreadable] achieved in the genital mucosa in the cells responsible for HIV transmission. This project will determine[unreadable] whether LC, DC, T cells and macrophages in the mouse genital tissue are silenced by siRNA-lipid[unreadable] complexes. If not, it will test in mice alternate cellular targeting strategies being developed in Project by Lieberman and[unreadable] the siRNA Manufacturing and Toxicology Core. Some of the questions to be answered include: How long do siRNAs reside in the vagina before[unreadable] uptake into cells? What is the duration of silencing in different cell types in situ? Does expression of the[unreadable] target gene affect the duration of silencing? How durable is protection from HSV-2 challenge after siRNA[unreadable] administration and how late after exposure can siRNAs be administered for post-exposure protection? How[unreadable] does menstrual variation affect uptake, gene silencing and protection in the HSV-2 model? Is there systemic[unreadable] exposure after topical administration? As siRNAs are optimized in the siRNA Manufacturing and Toxicology Core and Project by Lieberman as to sequence,[unreadable] chemical modification for stability and delivery, and formulation, their initial in vivo testing will be in this[unreadable] project. These results, with those from project by Lieberman, will be used to select lead candidates and design optimal[unreadable] primate experiments in Project by Veazey, where cost precludes testing too many variables.[unreadable]